Phase-Contrast Microscopy for Evaluating Developmental Mutants in Streptomyces

Abstract

Source: Bennett, J. A. et al. Visual and Microscopic Evaluation of Streptomyces Developmental Mutants. J. Vis. Exp. (2018)

This video demonstrates the use of phase-contrast microscopy to examine phenotypic differences between wild-type and mutant Streptomyces coelicolor. It outlines the steps involved in preparing and imaging aerial filaments to detect developmental defects in spore formation.

Protocol

1. Perform Phase-Contrast Microscopy

1. Sterilize tweezers by washing in 70% ethanol and then passing through a Bunsen burner flame to evaporate the ethanol. Allow to cool.
2. Sterilize coverslips by washing them in 70% ethanol and then allowing them to dry in a sterile Petri dish.
3. Prepare a coverslip lift of the bacterial growth to be examined.
   1. Pick up a sterile coverslip with sterile tweezers and place the coverslip on a mannitol soya flour (MS) agar plate where Streptomyces coelicolor bacterial growth is dense. Press on the back of coverslip gently with tweezers to ensure sufficient tr....

Results

Figure 1: Phase-contrast micrographs showing a representative aerial filament for Strept.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
50% Glycerol	Sigma-Aldrich	G5516
Immersion Oil (Type DF)	Cragille	16482
Sterile Water	GeneMate	G-3250-1L
100% Ethanol	Sigma-Aldrich	E7023
Soy Flour	Bob's Red Mill	1516C164
D-Mannitol	Sigma-Aldrich	M4125-1KG
Agar	Sigma-Aldrich	A1296-1KG
Glycerol 100%	VWR amresco life science	0854-1L
Petri dishes	Sigma-Aldrich	P5856-500EA
Cover slips #1.5	Thomas Scientific	64-0721
Slides	Carolina	63-2010
Phase-Contrast Microscope	Olympus	BX40
Forceps	Carolina Biological	624504
Bunsen Burner	Carolina Biological	706706
Clear nail polish	OPI	22001009000
Camera for Microscope	Olympus	DP72
Nitrile Examination Gloves (Med)	Bio Excell	71011002